Novel srna platform for inhibiting prokaryotic expression and use thereof

Abstract

The present disclosure relate to a composition for inhibiting a prokaryotic expression and a use thereof and, more specifically, to a composition for inhibiting an expression of Gram-positive bacteria, which includes an sRNA comprising an sRNA-derived Hfq binding site from prokaryotes and (ii) a region that forms a complementary bond with a target gene mRNA and an Hfq from prokaryotes, a method of producing same, and a use thereof. A synthetic sRNA according to the present disclosure and a composition comprising the sRNA for inhibiting a gene expression having an advantage of being able to control single and multiple target genes at a time, can effectively reduce the expression of the target gene without the conventional gene deletion process via the synthetic sRNA that controls a gene expression so as to be useful for the production of a recombinant microorganism, and are particularly useful for inhibiting a gene expression of Gram-positive bacteria. A recombinant Corynebacterium produced by the present disclosure is a recombinant microorganism capable of mass production of high value products in an ecofriendly and reproducible manner on a bio-basis by controlling microbial metabolism flow through the synthetic sRNA. The recombinant microorganism, which is a bio-based production system developed through the sRNA is useful because of being able to replace existing fossil fuels while resolving environmental problems due to the ever-increasing use of crude oil.

1 . Synthetic sRNA for inhibiting gene expression in a prokaryote, the synthetic sRNA comprising:
 (i) an Hfq binding site derived from sRNA of any one selected from the group consisting of sprX2, roxS, arnA, surA, ASdes, ASpks, AS1726, AS1890, Mcr1˜19, Mpr1˜21, B11, B55, C8, F6, G2, ncRv12659, fsrA, crcZ, SR1, 6S-1, ncr1175, ncr982, ncr1241, ncr1015, ncr1241, ncr1575, ncr952, ncr629, cgb_03605, cgb_00105, cgb_20715, IGR-1˜12, AS-1˜12, sgs2672, sgs3323, sg53618, sgs4453, sgs4827, sgs2746, sgs3903, sg54581, sgs5362, sgs5676, sgs6100, sgs6109, scr1906, scr2101, scr3261, scr3261, α3287, scr3558, scr3974, scr4677 and scr5676; and   (ii) a region forming a complementary bond with a target gene mRNA.   
     
     
         2 . The synthetic sRNA according to  claim 1 , wherein the region forming the complementary bond with the target gene mRNA entirely or partially forms a complementary bond with nucleic acid sequences corresponding to a start of a ribosome binding site of the target gene mRNA to an end of a gene-coding sequence. 
     
     
         3 . The synthetic sRNA according to  claim 1 , wherein the prokaryote is any one selected from the group consisting of  Escherichia coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, Pasteurella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter, Acinetobacter, Ralstonia, Agrobacterium, Rhizobium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium  and  Cyclobacterium.    
     
     
         4 . A nucleic acid encoding the sRNA according to  claim 1 . 
     
     
         5 . A recombinant prokaryote introduced with a replicable form of the nucleic acid according to  claim 4 . 
     
     
         6 . An expression vector comprising the nucleic acid encoding the sRNA according to  claim 1 . 
     
     
         7 . A recombinant prokaryote transformed with the expression vector according to  claim 6 . 
     
     
         8 . A nucleic acid comprising the nucleic acid according to  claim 4  and a nucleic acid encoding prokaryote-derived Hfq. 
     
     
         9 . The nucleic acid according to  claim 8 , wherein the prokaryote-derived Hfq is any one selected from the group consisting of  Escherichia coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, Pasteurella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter, Acinetobacter, Ralstonia, Agrobacterium, Rhizobium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium  and  Cyclobacterium.    
     
     
         10 . A recombinant prokaryote introduced with a replicable form of the nucleic acid according to  claim 8 . 
     
     
         11 . An expression vector comprising the nucleic acid according to  claim 4  and a nucleic acid encoding prokaryote-derived Hfq. 
     
     
         12 . A recombinant prokaryote introduced with an expression vector comprising a nucleic acid encoding the sRNA according to  claim 1  and a nucleic acid encoding prokaryote-derived Hfq, or introduced with a recombinant vector comprising an expression vector comprising a nucleic acid encoding the sRNA according to  claim 1  and a nucleic acid encoding prokaryote-derived Hfq. 
     
     
         13 . A method of inhibiting expression of a target gene in a prokaryote comprising culturing the recombinant prokaryote according to  claim 12  to inhibit mRNA of the target gene. 
     
     
         14 . A method of screening a gene targeted for deletion for production of a useful substance comprising:
 (a) inhibiting expression of at least one of genes present in a target strain for producing the useful substance and participating in a biosynthetic pathway of the useful substance using the method according to  claim 13 ; and   (b) selecting the gene, expression of which is inhibited, as the gene targeted for deletion for the production of the useful substance when a production yield of the useful substance is improved due to the inhibition of expression.   
     
     
         15 . A method of improving a strain for producing a useful substance comprising deleting a gene screened by the method according to  claim 14  or a combination of the screened gene to produce a recombinant strain.